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Human, but not rat, IRS1 targets to the plasma membrane in both human and rat primary adipocytes
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Health Sciences, Internal Medicine . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
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2007 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 363, no 3, 840-845 p.Article in journal (Refereed) Published
Abstract [en]

Adipocytes are primary targets for insulin control of metabolism. The activated insulin receptor phosphorylates insulin receptor substrate-1 (IRS1), which acts as a docking protein for downstream signal mediators. In the absence of insulin stimulation, IRS1 in rat adipocytes is intracellular but in human adipocytes IRS1 is constitutively targeted to the plasma membrane. Stimulation of adipocytes with insulin increased the amount of IRS1 at the plasma membrane 2-fold in human adipocytes, but >10-fold in rat adipocytes, with the same final amount of IRS1 at the plasma membrane in cells from both species. Cross-transfection of rat adipocytes with human IRS1, or human adipocytes with rat IRS1, demonstrated that the species difference was due to the IRS1 protein and not the cellular milieus or posttranslational modifications. Chimeric IRS1, consisting of the conserved N-terminus of rat IRS1 with the variable C-terminal of human IRS1, did not target the plasma membrane, indicating that subtle sequence differences direct human IRS1 to the plasma membrane.

Place, publisher, year, edition, pages
2007. Vol. 363, no 3, 840-845 p.
Keyword [en]
Insulin receptor substrate; Human; Rat; Insulin; Plasma membrane; Signaling; Transfection; Caveolae
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-14540DOI: 10.1016/j.bbrc.2007.09.065OAI: oai:DiVA.org:liu-14540DiVA: diva2:23675
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2017-12-13
In thesis
1. A molecular approach to insulin signalling and caveolae in primary adipocytes
Open this publication in new window or tab >>A molecular approach to insulin signalling and caveolae in primary adipocytes
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The prevalence of type II diabetes is increasing at an alarming rate due to the western world lifestyle. Type II diabetes is characterized by an insulin resistance distinguished by impaired glucose uptake in adipose and muscle tissues. The molecular mechanisms behind the insulin recistance and also the knowledge considering normal insulin signalling in fat cells, especially in humans, are still unclear.

Insulin receptor substrate (IRS) is known to be important for medating the insulin-induced signal from the insulin receptor into the cell. We developed and optimized a method for transfection of primary human adipocytes by electroporation. By recombinant expression of proteins, we found a proper IRS to be crucial for both mitogenic and metabolic signalling in human adipocytes. In human, but not rat, primary adipocytes we found IRS1 to be located at the plasma membrane in non-insulin stimulated cells. Insulin stimulation resulted in a two-fold increase of the amount of IRS1 at the plasma membrane in human cells, compared with a 12-fold increase in rat cells. By recombinant expression of IRS1 we found the species difference between human and rat IRS1 to depend on the IRS proteins and not on properties of the host cell.

The adipocytes function as an energy store, critical for maintaining the energy balance, and obesity strongly correlates with insulin resistance. The insulin sensitivity of the adipocytes with regard to the size of the cells was examined by separating small and large cells from the same subject. We found no increase of the GLUT4 translocation to the plasma membrane following insulin stimulation in the large cells, whereas there was a two-fold increase in the small cells. This finding supports the idea of a causal relationship between the enlarged fat cells and reduced insulin sensitivity found in obese subjects.

The insulin receptor is located and functional in a specific membrane structure, the caveola. The morphology of the caveola and the localization of the caveolar marker proteins caveolin-1 and -2 were examined. Caveolae were shown to be connected to the exterior by a narrow neck. Caveolin was found to be located at the neck region of caveolae, which imply importance of caveolin for maintaining and sequestering caveolae to the plasma membrane.

In conclusion, the transfection technique proved to be highly useful for molecular biological studies of insulin signal transduction and morphology in primary adipocytes.

Place, publisher, year, edition, pages
Institutionen för biomedicin och kirurgi, 2007. 63 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 977
Keyword
caveolae, insulin signalling, adipocytes
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-8960 (URN)91-85497-94-0 (ISBN)
Public defence
2007-01-19, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2013-09-10
2. Caveolae structure and importance in insulin action
Open this publication in new window or tab >>Caveolae structure and importance in insulin action
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type II diabetes is a disease characterized by chronic hyperglycaemia and abnormalities in lipid metabolism that affects approximately 5% of the population in the Western World. Caveolae are invaginations of the plasma membrane, described as 25-150 nm omega shaped structures, which are enriched in cholesterol, sphingolipids and the constituent protein caveolin. Caveolae have been shown to be involved in signal transduction, uptake over the plasma membrane and intracellular transport. By electron microscopy studies of cell membranes and biochemical analyses of isolated caveolae, we report that in rat adipocytes glucose transporter GLUT4 was translocated to caveolae in response to insulin. Insulin stimulation increased the amount of GLUT4 in the plasma membrane, but the ratio between GLUT4 in the planar and caveolae membrane remained constant. These findings indicate that caveolae are the locales for glucose uptake in the cell. We also report that the insulin receptor, independently of insulin stimulation, was localised in caveolae in human adipocytes. In these cells depletion of cholesterol destroyed the caveolae structure and the adipocytes became insulin resistant. Cholesterol depletion did not affect the insulinstimulated autophosphorylation of the insulin receptor nor the phosphorylation of the downstream IRS1. Further signalling to metabolic control or mitogenic control was inhibited, however. With transmission electron-, scanning electron- and fluorescence-microscopic techniques, we studied the ultrastructure and distribution of caveolae in the rat adipocyte. We found that caveolae can be divided into two subpopulations, small (<50 nm) and large (50-150 nm). The large caveolae are connected to the extracellular space via narrow necks and the orifices of caveolae were herein shown in primary adipocytes for the first time. Caveolin is located in the membrane proximal part of the small caveolae and to the neck in the large caveolae. The insulin receptor substrate IRS 1 was shown to be localized to caveolae in human adipocytes and to colocalize with the insulin receptor. In rat adipocytes, however, IRS1 was not localized to the plasma membrane in the absence of insulin stimulation. By transfection of rat adipocytes with human IRS1 we found that human IRS1 bound to the plasma membrane in the rat adipocyte, whereas the endogenous rat IRS1 did not. Taken together, caveolae seem to be closely involved in regulation of insulin action in the adipocyte.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2004. 53 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 875
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24065 (URN)3624 (Local ID)91-7373-851-4 (ISBN)3624 (Archive number)3624 (OAI)
Public defence
2004-12-10, Berzeliussalen, Hälsouniversitetet, Linköping, 09:00
Opponent
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-25Bibliographically approved

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Stenkula, Karin G.Thorn, HansFranck, NiclasHallin, ElisabethSauma, LilianStrålfors, PeterNyström, Fredrik H.

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